Alphaviruses are an important group of human and animal pathogens that are widely distributed on all continents. Until recently, the importance of the Old World alphaviruses in global human health was strongly underestimated. However, recent outbreaks of chikungunya virus (CHIKV) in India, Italy, East Africa's coastal islands and most recently on the Caribbean islands, South and Central America, Oceania have highlighted the risk posed by this virus. Our previous studies have unambiguously demonstrated that the Old World alphaviruses employ their nonstructural protein nsP2 to re-direct the cellular transcription coupled repair pathway for degradation of the catalytic subunit of the DNA- dependent RNA polymerase II, RPB1, and thus, to completely inhibit cellular transcription. Within a few hours post infection, virus-induced transcriptional shutoff makes the cells incapable of initiating an antiviral response and leads to development of cytopathic effect. Thus, the Old World alphavirus nsP2 is the major determinant of pathogenesis at the molecular and cellular levels. We have also demonstrated that the transcription inhibitory functions of nsP2 are determined not by its proteolytic activity, but by the ability of this protein to function as a helicase, and by its S-adenosyl-L- methionine-dependent RNA methyltransferase-like (SAM-like) domain. Two specific aims of the proposed research plan are focused on i) mechanistic understanding of the nsP2 helicase domain function in stalling cellular DNA-dependent RNA polymerase II and ii) on identification of cellular proteins that interact with the SAM-like domain of nsP2. We will also define the mechanistic role of the interactions of the latter domain in transcription inhibition. In our preliminary studies, we have developed a set of CHIKV variants with mutated nsP2, which are incapable of interfering with development of the innate immune response to CHIKV replication. These irreversible mutations had no detectable effect on the rates of CHIKV replication in cells defective in type I IFN signaling, but are cleared from IFN-competent cells without development of cytopathic effect. Therefore, in the third specific aim, we intend to assess the potential of these nsP2 mutants to improve the safety of the currently available experimental vaccine CHIKV 181/25.